Protein binding to akt2

ABSTRACT

A novel polypeptide useful in screening an insulin resistance improving agent and a carbohydrate metabolism improving agent, a polynucleotide coding for the aforementioned polypeptide, an expression vector comprising the aforementioned polynucleotide, and a cell transfected with the aforementioned expression vector are disclosed. The aforementioned polypeptide is a protein which is expressed in fat, and the activity of Akt2 is reduced in a fat cell in which the protein is highly expressed. A method for screening an insulin resistance improving agent and a carbohydrate metabolism improving agent using the aforementioned polypeptide, and a method for producing a pharmaceutical composition for insulin resistance improvement and carbohydrate metabolism improvement, which uses a substance obtained by said screening method as the active ingredient, are disclosed.

TECHNICAL FIELD

The present invention relates to a novel polypeptide which binds toAkt2, and a novel polynucleotide coding for said polypeptide, a vectorcomprising said polynucleotide, a transformed cell comprising saidvector and a method for screening a substance which inhibits binding ofthe aforementioned polypeptide with Akt2.

BACKGROUND OF THE INVENTION

Insulin is secreted from β cells of the pancreatic islets of Langerhansand reduces blood sugar level by mainly acting upon muscle, the liverand adipose and thereby incorporating blood sugar into cells to effectits storage and consumption. Diabetes mellitus is induced byinsufficient action of this insulin, and two types are present in itspatients, namely type 1 having a disorder of the production or secretionof insulin and type 2 having a difficulty in accelerating carbohydratemetabolism by insulin. The blood sugar level in both of these patientsbecomes higher than that in healthy person, but while insulin in bloodis absolutely insufficient in type 1, insulin resistance occurs in type2 in which incorporation or consumption of blood sugar by cells is notaccelerated in spite of the presence of insulin. The type 2 diabetesmellitus is a so-called lifestyle-related disease which is generated dueto overeating, lack of exercise, stress and the like causes in additionto the hereditary predisposition. Nowadays, patients of this type 2diabetes mellitus are rapidly increasing in advanced nations accompaniedby the increase of ingestion calories, and this type occupies 95% ofdiabetes mellitus patients in Japan. Accordingly, necessity isincreasing not only for a simple hypoglycemic drug as a therapeuticagent for diabetes mellitus but also on a study which aims at treatingtype 2 diabetes mellitus for the purpose of accelerating carbohydratemetabolism through the improvement of insulin resistance.

At present, insulin injections are prescribed for the treatment of type1 diabetes mellitus patients. On the other hand, in addition to theinsulin injections, sulfonylurea hypoglycemic agents (SU preparations)which prompts secretion of insulin by acting upon β cells of thepancreas and biguanide hypoglycemic agents which have actions toincrease sugar usage and inhibit gluconeogenesis by anaerobic glycolysisaction and to inhibit intestinal absorption of sugar, as well asa-glucosidase inhibitors which delay digestion and absorption ofsaccharides, are known as the hypoglycemic agents prescribed for type 2patients. Though these indirectly improve insulin resistance,thiazolidine derivatives have been used in recent years as agents whichdirectly improve insulin resistance. Its action is to accelerateincorporation of glucose into cells and use of glucose in cells. It isshown that the thiazolidine derivatives act as an agonist of peroxisomeproliferator responding activated receptor gamma (PPARγ) (cf. Non-patentreference 1). However, it is known that the thiazolidinediones not onlyimprove insulin resistance but also have a side effect to induce edema(cf. Non-patent reference 2, Non-patent reference 3). Since thisinduction of edema is a serious side effect which results in cardiachypertrophy, more useful target molecule for drug development than thePPARγ is in demand for the improvement of insulin resistance.

The signal of insulin action is transferred into cells via an insulinreceptor on the cell membrane. Two pathways of the first and second arepresent in this insulin action pathway (cf. Non-patent reference 4). Inthe first pathway, the signal is transferred in order from the activatedinsulin receptor to Akt1 (PKBα) or Akt2 (PKBβ), or PKCγ or PKCζ, viaIRS-1, IRS-2, PI 3 kinase and PDK 1, and incorporation of sugar from theextracellular moiety is accelerated as the result by translocating aglucose transporter GLUT 4 existing inside the cell onto the cellmembrane (cf. Non-patent reference 5). On the other hand, in the secondpathway, the signal is transferred from the insulin receptor to CrK II,C3G and TC 10 in that order via c-Cbl and CAP, and incorporation ofsugar by GLUT 4 is accelerated as the result (cf. Non-patent reference6). However, there are portions still unclear regarding details of theseinsulin signal transduction pathways, and it is not clear particularlyabout the mechanism which finally mediates acceleration of sugarincorporation of cells by these signals via the glucose transporter.

Akt2 is present in the aforementioned insulin signal first pathway andactivated by undergoing phosphorylation by insulin stimulation viaPDK 1. The activated Akt2 transfers the signal as a kinase byphosphorylating a protein as its substrate. It has been reported that ahomo-knockout mouse in which a gene coding for the Akt2 protein wasartificially deleted shows a type 2 diabetes-like phenotype due toreduced insulin sensitivity mainly in muscle and the liver. Based onthese facts, it has been considered that Akt2 is a signal mediatingfactor which functions in incorporating sugar into cells in response tothe insulin signal, and its functional inhibition induces insulinresistance by partial interception of the insulin signal transduction(cf. Non-patent reference 7).

(Non-patent Reference 1)

“The Journal of Biological Chemistry”, (USA), 1995, vol. 270, pp.12953-12956

(Non-patent Reference 2)

“Diabetes Frontier”, (USA), 1999, vol. 10, pp. 811-818

(Non-patent Reference 3)

“Diabetes Frontier”, (USA), 1999, vol. 10, pp. 819-824

(Non-patent Reference 4)

“The Journal of Clinical Investigation”, (USA), 2000, vol. 106, no. 2,pp. 165-169

(Non-patent Reference 5)

“The Journal of Biological Chemistry”, (USA), 1999, vol. 274, no. 4, pp.1865-1868

(Non-patent Reference 6)

“Nature”, (England), 2001, vol. 410, no. 6831, pp. 944-948

(Non-patent Reference 7)

“Science”, (USA), 2002, vol. 292, no. 2, pp. 1728-1731

DISCLOSURE OF THE INVENTION

Based on the aforementioned information, the present inventorsconsidered that insulin resistance may be improved when function of Akt2can be increased. It was considered that this object may be achieved byincreasing activity of the Akt2 itself, or by regulating the activity ofa newly identified intracellular factor which binds to Akt2 and therebycontrols its activity. However, since Akt2 is a kinase, it is difficultto regulate its enzyme activity toward increasing direction by a drug.Accordingly, a protein which binds to Akt2 was identified by a yeast twohybrid system. As a result, it was successful in cloning a mouse derivedcDNA of a novel nucleotide sequence coding for a protein AKBP 2 (Akt2Binding Protein 2) which binds to Akt2. Also, since expressed amount ofthis protein was considerably increased in muscle and adipose of modelmice of diabetes mellitus in comparison with normal individuals, it wasfound that this protein is a causal factor of the morbid state ofdiabetes mellitus. In addition, by succeeding in cloning a humanorthologue human AKBP 2 gene, it was found that said gene is expressedin fat cells as an insulin response tissue and that human AKBP 2 alsobinds to Akt2 as the case of mouse AKBP 2. In addition to this, bydetecting that kinase activity of Akt2 is reduced by overexpression ofmouse AKBP 2, it was found that insulin resistance is induced by theinterception of insulin signal by AKBP 2, that is, insulin resistance isimproved by-inhibiting binding of AKBP 2 with Akt2. Accordingly, ascreening system for an insulin resistance improving agent and/or acarbohydrate metabolism improving agent was constructed making use ofthe interaction of AKBP 2 with Akt2.

As these results, the present invention was accomplished by providing anovel polypeptide useful in screening an insulin resistance improvingagent and/or a carbohydrate metabolism improving agent, a polynucleotidecoding for the aforementioned polypeptide, an expression vectorcomprising the aforementioned polynucleotide, a cell transformed withthe aforementioned expression vector, a method for screening an insulinresistance improving agent and/or a carbohydrate metabolism improvingagent, and a method for producing a pharmaceutical composition forinsulin resistance improvement and/or carbohydrate metabolismimprovement.

That is, the present invention relates to

[1] a polypeptide which comprises the amino acid sequence represented bySEQ ID NO:2 or SEQ ID NO:4, or an amino acid sequence in which from 1 to10 amino acids are deleted, substituted and/or inserted in the aminoacid sequence represented by SEQ ID NO:2 or SEQ ID NO:4, and which bindsto Akt2,

[2] a polypeptide consisting of the amino acid sequence represented bySEQ ID NO:2 or SEQ ID NO:4,

[3] a polynucleotide coding for the polypeptide described in [1] or [2],

[4] an expression vector comprising the polynucleotide described in [3],

[5] a cell transformed with the expression vector described in [4],

[6] a method for screening a substance which inhibits binding of apolypeptide described in claim 1 or claim 2 or a polypeptide consistingof an amino acid sequence having a homology of 90% or more with theamino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4 and whichbinds to Akt2, with Akt2, which comprises

allowing (1) the aforementioned polypeptide or a cell expressing theaforementioned polypeptide, to contact (2) a substance to be tested,

measuring binding of said polypeptide with Akt2, and

selecting a substance which inhibits the aforementioned binding,

[7] the screening method described in [6], wherein the bindinginhibiting substance is an insulin resistance improving agent and/or acarbohydrate metabolism improving agent,

[8] the screening method described in [6] or [7], wherein the step ofmeasuring binding of (1) the polypeptide described in [1] or [2] or apolypeptide consisting of an amino acid sequence having a homology of90% or more with the amino acid sequence represented by SEQ ID NO:2 orSEQ ID NO:4, and which binds to Akt2, to (2) Akt2 is a step of measuringa change in Akt2 based on the change in the aforementioned binding, and

[9] a method for producing a pharmaceutical composition for insulinresistance improvement and/or carbohydrate metabolism improvement, whichcomprises

carrying out screening using the screening method described in [6] to[8], and

preparing a pharmaceutical preparation.

Virtually nothing is known about the sequences identical to thepolypeptides and polynucleotides of the present invention described inSEQ ID NOs:1 to 4. Though sequences having homology with thepolynucleotides of the present invention have been reported in asequence data base GenBank as accession numbers AX714043, BC042155 andBC049110 after the priority date of this application, this is merely adisclosure of sequences and there is no description on theirillustrative use. Also, a sequence data base GenPept carries, as anaccession number AK056090, a polypeptide consisting of an amino acidsequence in which 68 amino acids of the amino acid sequence representedby SEQ ID NO:4 as one of the polypeptides of the present invention aredeleted, and as an accession number AK019105, a polypeptide consistingof an amino acid sequence in which 228 amino acids of the amino acidsequence represented by SEQ ID NO:2 as one of the polypeptides of thepresent invention are deleted and 13 amino acids of the same aresubstituted. However, there is no information that these polypeptideswere actually prepared, and there is no information on how to preparethem. In addition, illustrative use of said polypeptides is notdescribed either. The present inventors have found the polypeptides andpolynucleotides of the present invention for the first time and revealedfor the first time that overexpression of the protein and increase ofits binding to Akt2 are causal factors of the morbid state of diabetesmellitus. In addition, the screening methods of the present invention bymaking use of the binding of the polypeptide of the present inventionwith Akt2 are methods provided for the first time by the presentinventors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing expression of AKBP2 in cultured cells. Lanes 1and 3 indicate molecular weight markers, and lane 2 an empty vector andlane 4 a case of introducing pcDNA-AKBP2.

FIG. 2 (1) is a graph showing comparison of AKBP2 expression level inadipose of normal mice C57BL/6J loaded with a normal feed or a high fatfeed. Vertical axis of the drawing shows relative expression level inmouse fat. The white bar shows when the normal feed was loaded, and theblack bar when the high fat feed was loaded.

The (2) is a graph showing comparison of AKBP2 expression level inmuscle of the normal mice C57BL/6J loaded with a normal feed or a highfat feed. Vertical axis of the drawing shows relative expression levelin mouse muscle. The white bar shows when the normal feed was loaded,and the black bar when the high fat feed was loaded.

The (3) is a graph showing comparison of AKBP2 expression level inadipose of the normal mice C57BL/6J and diabetes mellitus model miceKKA^(y)/Ta. Vertical axis of the drawing shows relative expression levelin mouse adipose. The white bar shows a result of the normal miceC57BL/6J, and the lined bar that of the diabetes mellitus model miceKKA^(y)/Ta.

The (4) is a graph showing comparison of AKBP2 expression level inmuscles of the normal mice C57BL/6J and the diabetes mellitus model miceKKA^(y)/Ta. Vertical axis of the drawing shows relative expression levelin mouse muscle. The white bar shows a result of the normal miceC57BL/6J, and the lined bar that of the diabetes mellitus model miceKKA^(y)/Ta.

Expression level in normal feed C57BL/6J is shown as 1 in the comparisonin (1) and (2), and expression level in C57BL/6J is shown as 1 in thecomparison in (3) and (4), respectively.

FIG. 3 is a graph showing influence of mouse AKBP2 overexpression inNIH3T3 L1 fat cells upon Akt2 enzyme activity. Vertical axis of thedrawing shows relative activity, and the value of the enzyme activity inthe cells infected with a control virus under the condition of noinsulin stimulus is shown as 1. Horizontal axis of the drawing showsinsulin stimulation period (minute).

BEST MODE FOR CARRYING OUT THE INVENTION

The following describes the present invention in detail.

<Polypeptide of the Invention>

Included in the polypeptide of the present invention are

-   (1) a polypeptide consisting of the amino acid sequence represented    by SEQ ID NO:2 or SEQ ID NO:4; and-   (2) i) a polypeptide which comprises the amino acid sequence    represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt2,    or ii) a polypeptide which comprises an amino acid sequence in which    from 1 to 10 (preferably from 1 to 7, more preferably from 1 to 5,    further preferably from 1 to 3) amino acids are deleted, substituted    and/or inserted in the amino acid sequence represented by SEQ ID    NO:2 or SEQ ID NO:4 and which binds to Akt2 (to be referred to as    functionally equivalent variant hereinafter).

Those which reduce kinase activity of Akt2 by binding to Akt2 areparticularly desirable as the polypeptides of the present invention.

Also, the polypeptides of the present invention are not particularlylimited to the human and mouse derived polypeptides as long as they comeunder either of the aforementioned (1) and (2), and those which arederived from other vertebrates (e.g., rat, rabbit, horse, sheep, dog,monkey, cat, bear, pig, domestic fowl and the like) are also includedtherein. In addition, they are not limited to natural polypeptides andartificially produced mutants are also included therein, as long as theycome under either of the aforementioned (1) and (2).

The term “binds to Akt2” means that a polypeptide binds to Akt2(preferably human Akt2, more preferably the polypeptide encoded by theGenBank accession number M95936), and whether or not it “binds” may beverified by the following methods.

A partial or the entire length of a polypeptide to be examined forwhether or not it binds, or a partial or the entire length of apolypeptide to be examined fused with a tag (e.g., GST, Flag, His or thelike), is expressed in a cell. As the aforementioned cell, a cell whichresponds to insulin is desirable, and more illustratively, fat cell,hepatocyte or a skeletal muscle-derived cell is desirable. Akt2 proteinand a protein which binds thereto may be concentrated from theaforementioned cell by immunoprecipitation using an anti-Akt2 antibody.Whether or not the polypeptide to be examined binds to Akt2 may beverified by separating concentrated solution of the thus obtained Akt2and its binding protein by polyacrylamide gel electrophoresis through aconventionally known method and then carrying out western blotting usingan antibody. Regarding the antibody to be used in this case, an antibodyfor the polypeptide to be examined or a polypeptide to be examinedprepared based on its partial sequence, or an antibody which recognizesthe aforementioned tag may be used.

In addition, binding of the polypeptide to be examined with apolypeptide can also be detected by combining a western blotting similarto the aforementioned one with an in vitro pull down method (H.Matsushime et al., Jikken Kogaku (Experimental Engineering), Vol. 113,No. 6, p. 528, 1994) which uses an extract of cells in which thepolypeptide to be examined is expressed, or a mixed solution of proteinsprepared by in vitro transcription and translation, and Akt2 proteinpurified by attaching a tag (e.g., GST or the like). Preferably, thebinding may be detected using a mixed solution of proteins prepared bydirectly carrying out in vitro transcription and translation of theprotein to be examined as shown in Example 6, from a plasmid for use inthe expression of the protein to be examined (e.g., the plasmid forAKBP2 protein expression prepared in Example 1(5)) using an in vitrotranslation kit (e.g., TNT Kit (Promega)). More preferably, binding ofthe polypeptide to be examined to Akt2 may be detected by the methoddescribed in Example 6. The term “to reduce kinase activity of Akt2”means that the kinase activity possessed by Akt2 is reduced through thebinding of the polypeptide to be examined to Akt2. Whether or not the“kinase activity is reduced” may be verified by the following method.

It is known that the kinase activity of Akt2 is accelerated when the473rd serine (Ser 473) or the 308th threonine (Thr 308) in the moleculeis phosphorylated (Biochem. J., 1998, 335 (1-13)). Making use of this,the presence or absence of Akt2 activity may be detected by detectingphosphorylated condition of the Ser 473 or Thr 308 of Akt2 by a westernblotting which uses an antibody capable of specifically reacting withthese phosphorylated residues (e.g. anti-phosphoSer antibody or thelike). More illustratively, phosphorylation of Akt2, namely the presenceor absence of Akt2 activity, may be detected by lysing cells (a cellwhich responds to insulin is desirable, and more illustratively, fatcell, hepatocyte or a skeletal muscle-derived cell is desirable) inwhich a part or the entire length of the polypeptide to be examined isexpressed, and using this as the sample, carrying out western blotting,spot western blotting or the like method which uses the anti-phosphoSerantibody. Preferably, this may be detected by the method of Example 7.When reduction of the phosphorylation of Akt2 (namely activation ofAkt2) was observed in this detection system by the use of a sampleobtained from a cell in which the polypeptide to be examined wasexpressed, in comparison with a cell in which the polypeptide to beexamined was not expressed, it may be judged that the polypeptide to beexamined “reduces kinase activity of Akt2”.

In addition, whether or not it “reduces kinase activity of Akt2” canalso be verified by an in vitro kinase assay method in which uptake ofradioactive phosphoric acid based on a substrate is measured when ahistone H2B, a GSK-3 fusion protein or the like is used as the substrateof Akt2 and allowed to react with an immune precipitate of Akt2.Illustratively, Akt2 protein may be concentrated from an extract ofcells (a cell which respond to insulin is desirable, and moreillustratively, fat cell, hepatocyte or a skeletal muscle-derived cellis desirable) in which a part or the entire length of the polypeptide tobe examined is expressed, by immunoprecipitation using an anti-Akt2antibody. By mixing a substrate of Akt2, such as GST-crosstide (GSTfusion protein of GSK3-beta as a physiological substrate of Akt), withconcentrated Akt2 protein, kinase activity of Akt2 may be measured-anddetermined using phosphorylation of the substrate as the index.Preferably, this may be measured by the method described in Example 7.When reduction of the phosphorylation of the substrate was observed inthis measuring system by the use of a sample obtained from a cell inwhich the polypeptide to be examined was expressed, in comparison with acell in which the polypeptide to be examined was not expressed, it maybe judged that the polypeptide to be examined “reduces kinase activityof Akt2”.

<Polynucleotide of the Invention>

The polynucleotide of the present invention may be derived from anyspecies, as long as it encodes the polypeptide of the present invention,namely a polypeptide represented by the amino acid sequence described inSEQ ID NO:2 or SEQ ID NO:4, or a functionally equivalent variantthereof. Preferred is a polynucleotide consisting of a nucleotidesequence coding for the amino acid sequence described in SEQ ID NO:2 orSEQ ID NO:4, and further preferred is the nucleotide sequence describedin SEQ ID NO:1 or SEQ ID NO:3. In this connection, both of DNA and RNAare included in the “polynucleotide” according to this description.

All kinds of mutants may be included in the polynucleotide of thepresent invention, as long as they encode the polypeptide of the presentinvention. More illustratively, it can include allele mutants which arepresent in the natural world, mutants which are not present in thenatural world, and mutants which have deletion, substitution, additionand insertion. The aforementioned mutation sometimes occur by naturalmutation, but it can also be effected by carrying out artificialmodification. All mutant genes coding for the aforementioned polypeptideof the present invention are included in the present invention,regardless of the cause and means of mutation of the aforementionedpolypeptide. As the aforementioned artificial means leading to thepreparation of mutants, for example, in addition to genetic engineeringtechniques such as base-specific substitution method (Methods inEnzymology, (1987), 154, 350, 367-382) and the like, chemical synthesismeans such as phosphotriester method, phosphoamidide method and the like(Science, 150, 178, 1968) may be cited. By their combination, it ispossible to obtain a DNA accompanied by the desired base substitution.Alternatively, it is possible to generate a substitution in anonspecific base in the DNA molecule by the repetition of PCR or byallowing manganese ion or the like to be present in the reactionsolution.

The polynucleotide and polypeptide of the present invention may beeasily produced and obtained by general genetic-engineering techniquesbased on the sequence information disclosed by the present invention.

The polynucleotide coding for the polypeptide of the present inventionmay be prepared for example in the following manner, but it may beprepared not only by this method but also by conventionally knownoperations “Molecular Cloning, Sambrook, J. et al., Cold Spring HarborLaboratory Press, 1989” and the like.

For example, (1) a method which uses PCR, (2) general geneticengineering techniques (namely a method in which a transformantcomprising desired amino acids is selected from transformantstransformed with a cDNA library), (3) a chemical synthesis method or thelike may be cited. Each production method may be carried out in the samemanner as described in WO 01/34785.

By the method which uses PCR, the polynucleotide described in thisdescription may be produced for example by the procedure described inthe “Mode for Carrying Out the Invention”, 1) Production method ofprotein gene, a) First production method, of the aforementioned patentreference. Regarding the “human cell or tissue having the ability toproduce the protein of the present invention” in said description,adopose cells can for example be cited. Total mRNA is extracted fromhuman or murine adipose cells. Next, a first-strand cDNA may besynthesized by subjecting this mRNA-to a reverse transcriptase reactionin the presence of random primers or oligo dT primers. Thepolynucleotide of the present invention or a part thereof may beobtained by subjecting the thus obtained first-strand cDNA to polymerasechain reaction (PCR) using two kinds of primers interposing a partialregion of the gene of interest. More illustratively, the polynucleotideof the present invention may be produced for example by the methodsdescribed in Example 1 and Example 4.

By the method which uses general genetic engineering techniques, thepolynucleotide coding for the polypeptide of the present invention maybe produced for example by the procedure described in the “Mode forCarrying Out the Invention”, 1) Production method of protein gene, b)Second production method, of the aforementioned patent reference.

By the method which uses chemical synthesis, the polynucleotide codingfor the polypeptide of the present invention may be produced for exampleby the procedure described in the “Mode for Carrying Out theInvention”, 1) Production method of protein gene, c) Third productionmethod, d) Fourth production method, of the aforementioned patentreference.

By making use of partial or entire nucleotide sequence of the thusobtained polynucleotide of the present invention, expression level ofthe polynucleotide of the present invention in each individual orvarious tissues may be specifically detected.

Examples of such a detection method include RT-PCR (reversetranscribed-polymerase chain reaction), northern blotting analysis, insitu hybridization and the like methods.

<Production Methods of the Expression Vector, Cell and Polypeptide ofthe Invention>

Also included in the present invention is a method for producing thepolypeptide of the present invention, which is characterized in that thetransformed cell of the present invention is cultured.

The polynucleotide coding for the polypeptide of the present invention,obtained in the aforementioned manner, may be used for expressing thepolypeptide of the present invention in a test tube or in a test cell byconnecting it to the downstream of an appropriate promoter, byconventionally known methods described in “Molecular Cloning, Sambrook,J. et al., Cold Spring Harbor Laboratory Press, 1989” and the like.

Illustratively, by adding a polynucleotide containing a specifiedpromoter sequence to upstream of the initiation codon for thepolypeptide of the present invention obtained in the aforementionedmanner, it is possible to effect expression of the polypeptide of thepresent invention by cell-free system transcription and translation ofthe gene using this as the template.

Alternatively, expression of the polypeptide of the present invention ina cell becomes possible when the aforementioned polynucleotide codingfor the polypeptide of the present invention is integrated into anappropriate vector plasmid and introduced in the form of plasmid into ahost cell. Alternatively, a cell in which such a construction isintegrated into chromosomal DNA may be prepared and used. Moreillustratively, an isolated fragment containing a polynucleotide cantransform host cells of eukaryotes and prokaryotes by again integratinginto an appropriate vector plasmid. Furthermore, it is possible toeffect expression of the polypeptide of the present invention inrespective host cells by introducing an appropriate promoter and asequence concerned in the gene expression into these vectors. The hostcell is not particularly limited, as long as it can detect expression ofthe polypeptide of the present invention at the mRNA level or proteinlevel. It is most desirable to use a fat-derived cell or muscle-derivedcell as the host cell, in which endogenous Akt2 is abundantly present.

The method for expressing a gene by transforming a host cell may becarried out, for example, by the method described in the “Mode forCarrying Out the Invention”, 2) Method for producing the vector of thepresent invention, the host cell of the present invention and therecombinant protein of the present invention, of the aforementionedpatent reference. The expression vector is not particularly limited, aslong as it contains a desired polynucleotide. An example thereof is anexpression vector obtained by inserting the desired polynucleotide intoa conventionally known expression vector optionally selected in responseto the host cell to be used. The cell of the present invention may beobtained, for example, by transfecting a desired host cell with theaforementioned expression vector. More illustratively, an expressionvector for a desired protein may be obtained, for example, byintegrating a desired polynucleotide into an expression vector formammal cell, pcDNA3.1, as described in Example 2, and the transformedcell of the present invention may be produced by incorporating saidexpression vector into the 293 cell using the calcium phosphate method.

The desired transformed cell obtained in the above may be cultured inaccordance with a general method, and the desired protein is produced bysaid culturing. Regarding the medium to be used in said culturing,various generally used kinds may be optionally selected in response tothe employed host cell. For example, in the case of the aforementioned293 cell, Dulbecco's modified Eagle's minimum essential medium (DMEM)supplemented with serum component (e.g., fetal bovine serum (FBS) or thelike), to which G418 was further added, may be used. As the transformedcell of the present invention, a cell expressing the polypeptide of thepresent invention is desirable.

By culturing the cell of the present invention, the polypeptide of thepresent invention produced in the cell may be detected, determined andalso purified. For example, it is possible to detect and purify thepolypeptide of the present invention by western blotting orimmunoprecipitation using an antibody which binds to the polypeptide ofthe present invention. Alternatively, by expressing the polypeptide ofthe present invention as a fusion protein with appropriate tag protein(e.g.,. glutathione-S-transferase (GST), protein A, β-galactosidase,maltose-binding protein (MBP) or the like), the polypeptide of thepresent invention may be detected by western blotting orimmunoprecipitation using an antibody specific for such a tag proteinsand purified making use of the tag protein. More illustratively, it maybe purified making use of a tag protein in the following manner.

The polypeptide of the present invention (e.g., the polypeptiderepresented by SEQ ID NO:2 or SEQ ID NO:4) may be obtained byintegrating the polynucleotide of the present invention (e.g., thepolynucleotide represented by SEQ ID NO:1 or SEQ ID NO:3) for exampleinto a His tag fusing vector, more illustratively for example into thepcDNA3.1/V5-His-TOPO (Invitrogen) or the like described in Example 1, toeffect its expression in a cultured cell, purifying it using His tag,and then removing the tag moiety. For example, the mouse or human AKBP2expression plasmid prepared in Example 1 or Example 5 usingpcDNA3.1/V5-His-TOPO is designed in such a manner that V5 and His tagare added to the C-terminus of AKBP2 in both cases. Accordingly, theAKBP2 protein may be purified from the AKBP2-expressed cultured cellsshown in Example 2 or Example 5, using the His tag. Illustratively, theAKBP2 protein fused with His tag may be isolated from an extract ofdisrupted cells by binding it to Ni²⁺-NTA-Agarose (Funakoshi) andcentrifuging the product, in accordance with known methods (Jikken Igaku(Experimental Medicine) Supplement, Tanpakushitsu-no Bunshikan SogosayoJikkenhou (Experimentation on Intermolecular Interaction of Protein),page 32, 1996, Nakahara et al.). More illustratively, cells expressingthe polypeptide of the present invention cultured using a culture flasks(e.g., Petri dish of 10 cm in diameter) are scratched off after addingan appropriate volume (e.g., 1 ml) of a buffer solution and thencentrifuged at 1,5000 rpm for 5 minutes, and an appropriate amount(e.g., 50 μM) of Ni²⁺-NTA-Agarose substituted by an appropriate buffersolution is added to the thus separated supernatant and thoroughly mixed(e.g., 10 minutes or more of stirring using a rotator). Next, thesupernatant is separated and removed by centrifugation (e.g., 2,000 rpmfor 2 minutes) and again centrifuged by adding an appropriate amount(e.g., 0.5 ml) of a buffer solution adjusted to pH 6.8, therebyeffecting washing. After repeating this 3 times, an appropriate amount(e.g., 50 μl) of 100 mM EDTA is added, the mixture is allowed to standfor 10 minutes and then the supernatant is recovered, thereby enablingpurification of the released polypeptide of the present invention. Asthe aforementioned buffer solution, a buffer solution B (8 M urea, 0.1 MNa₂HPO₄, 0.1 M NaH₂PO₄, 0.01 M Tris-HCl pH 8.0) can for example be used.The His tag in the purified protein molecule may be removed from themolecule, for example, by designing in such a manner that His tag isfused to the N-terminal side and using TAGZyme System (Qiagen).

Alternatively, as occasion demands, it may be purified by a method whichdoes not use a tag protein, for example, by various separationoperations making use of the physical properties and chemical propertiesof the protein consisting of the polypeptide of the present invention.Illustratively, use of ultrafiltration, centrifugation, gel filtration,adsorption chromatography, ion exchange chromatography, affinitychromatography and high performance liquid chromatography may beexemplified.

The polypeptide of the present invention may be produced by generalchemical synthesis in accordance-with the amino acid sequenceinformation shown in SEQ ID NO:2 or SEQ ID NO:4. Illustratively, liquidphase and solid phase peptide synthesis methods are included. Itssynthesis may be carried out by successively binding one amino acidafter another, or by synthesizing a peptide fragment comprising severalamino acids and the binding it. The polypeptide of the present inventionobtained by these means may be purified in accordance with theaforementioned various methods.

<Inspection Method of Diabetes Mellitus>

By the use of a probe which hybridizes with the polynucleotide of thepresent invention under a stringent condition, expressed amount of apolynucleotide coding for the polypeptide of the present invention maybe examined, and diagnosis of diabetes mellitus can be carried out usingincrease of the expressed amount (preferably the expressed amount in thefat tissue) as the index. In the inspection method of diabetes mellitus,the term “stringent condition” means a condition under which nonspecificbinding does not occur, and illustratively, it means a condition inwhich 0.1×SSC (saline-sodium citrate buffer) solution containing 0.1%sodium lauryl sulfate (SDS) is used and the temperature is 65° C. As theprobe, a DNA of at least 15 bp in chain length and having at least apart of or entire sequence (or a complementary sequence thereof) of thepolynucleotide of the present invention is used.

According to the method for detecting diabetes mellitus, whether or notthe subject is diabetes mellitus may be detected by allowing theaforementioned probes to contact a sample to be tested, and analyzingthe bonded product of a polynucleotide coding for the polypeptide of thepresent invention (e.g., mRNA or cDNA derived therefrom) and theaforementioned probe by a conventionally known analyzing method (e.g.,northern blotting). In addition, the expression quantity can also beanalyzed by applying the aforementioned probe to a DNA tip. When theamount of the aforementioned bonded product, namely the amount of apolynucleotide coding for the polypeptide of the present invention, isincreased in comparison with healthy parsons, it may be judged that thesubject is diabetes mellitus.

As a method for measuring expressed level of the polynucleotide of thepresent invention, it is possible to employ methods in which theexpressed level is measured by detecting the polypeptide of the presentinvention. Examples of the inspection method to be used include westernblotting, immunoprecipitation, ELISA and the like methods, making use ofan antibody which binds a sample to be tested to the polypeptide of thepresent invention, or an antibody which specifically binds to thepolypeptide of the present invention. In determining the amount of thepolypeptide of the present invention contained in the sample to betested, the polypeptide of the present invention may be used as thestandard amount. In addition, the polypeptide of the present inventionis useful for preparing an antibody which binds to the polypeptide ofthe present invention. When the amount of the polypeptide of the presentinvention is increased in comparison with healthy parsons, it may bejudged that the subject is diabetes mellitus.

<Screening Method of the Invention>

By using (1) the polypeptide of the present invention, (2) a polypeptideconsisting of an amino acid sequence having a homology of 90% or morewith the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4and which binds to Akt2 (to be referred to as homologous polypeptidehereinafter), or (3) a polypeptide as a protein encoded by apolynucleotide which hybridizes with the polynucleotide having thenucleotide sequence represented by SEQ ID NO:1 or SEQ ID NO:3 under astringent condition and which binds to Akt2 (to be referred to ashybridize polypeptide hereinafter), a method for screening a substancehaving an insulin resistance improving action and/or a substance havingcarbohydrate metabolism improving action (namely diabetes mellitusimproving agents) may be constructed making use of the interaction ofthe aforementioned polypeptides (namely the polypeptide of the presentinvention, the homologous polypeptide and the hybridize polypeptide)with Akt2 kinase. The polypeptide of the present invention, theaforementioned homologous polypeptide and the aforementioned hybridizepolypeptide are generally referred to as a polypeptide for the screeningof the present invention.

The homologous polypeptide according to this description is notparticularly limited, as long as it is a polypeptide consisting of anamino acid sequence having a homology of 90% or more with the amino acidsequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds toAkt2, but regarding the amino acid sequence represented by SEQ ID NO:2or SEQ ID NO:4, a polypeptide consisting of an amino acid sequencehaving a homology of preferably 95% or more, more preferably 98% ormore, is desirable.

In this connection, the aforementioned term “homology” as used in thisdescription means a value (Identities) obtained using parametersprepared as default by Clustal program (Higgins and Sharp, Gene, 73,237-244, 1998; Thompson et al., Nucl. Acids Res., 22, 4673-4680, 1994)retrieval. The aforementioned parameters are as follows. As MultipleAlignment Parameters, Gap Penalty 15.00, Gap Length Penalty 6.66, DelayDivergent Seqs (%) 30 and DNA Transition Weight 0.50, and as PairwiseAlignment Parameters, Gap Penalty 15.00 and Gap Length Penalty 6.66, bySlow-Accurate.

Regarding the “stringent condition” for the hybridize polypeptide ofthis description under which a polynucleotide coding for the hybridizepolypeptide of this description hybridizes with the polynucleotidehaving the nucleotide sequence represented by SEQ ID NO:1 or SEQ IDNO:3, it is a condition of “5× SSPE, 5× Denhard's solution, 0.5% SDS,40% formamide, 200 μg/ml salmon sperm DNA, and 37° C. overnight” as thecondition for hybridization, and a condition of “5× SSPE, 5× Denhard'ssolution, 0.5% SDS, 50% formamide, 200 μg/ml salmon sperm DNA, and 42°C. overnight” as more strict condition. Also, the condition for washingis “5×SSC, 1% SDS and 42° C.” as a mild condition, “0.5×SSC, 0.1% SDSand 42° C.” as a normal condition, and “0.2×SSC, 0.1% SDS and 65° C.” asa more strict condition.

Also included in the screening method of the present invention is amethod for screening a substance which inhibits binding of theaforementioned polypeptide with Akt2, characterized in that it comprisesa step of allowing the polypeptide for screening of the presentinvention or a cell expressing the polypeptide for screening of thepresent invention or to contact a substance to be tested, a step ofmeasuring binding of said polypeptide with Akt2, and a step of selectinga substance which inhibits the aforementioned binding. The cellsexpressing the polypeptide for screening of the present invention may beeither cells transformed with an expression vector containing apolynucleotide coding for the polypeptide for screening of the presentinvention or a naturally existing cells expressing the polypeptide ofthe present invention, but a transformed cells are desirable.

Since the AKBP2 as one of the polypeptides for screening of the presentinvention binds to Akt2, its expression is reduced in diabetes mellitusmodel mice, and the Akt2 activity is reduced in fat cells in which mouseAKBP2 was overexpressed, it was found that the polypeptide of thepresent invention negatively controls the insulin signal via its bindingto Akt2. Thus, an insulin resistance improving agent and/or acarbohydrate metabolism improving agent may be screened by theaforementioned screening method.

In the aforementioned screening methods, the step of measuring bindingof the polypeptide for screening of the present invention or cellsexpressing the polypeptide for screening of the present invention withAkt2 may be carried out by directly detecting binding of the polypeptidefor screening of the present invention with Akt2, or can also be carriedout by measuring a change of Akt2 caused by a change of theaforementioned binding.

Though not particularly limited, examples of the substance to be testedwhich may be used in the screening methods of the present inventioninclude commercially available compounds (including peptides), variousconventionally known compounds registered in chemical files (includingpeptides), a group of compounds obtained by the combinatorial chemistrytechniques (N. K. Terrett, M. Gardner, D. W. Gordon, R. J. Kobylecki andJ. Steele, Tetrahedron, 51, 8135-73, (1995)), culture supernatants ofmicroorganisms, natural components derived from plants and marineorganisms, animal tissue extracts, or compounds (including peptides)obtained by chemically or biologically modifying a compound selected bythe screening methods of the present invention.

The aforementioned screening methods are not limited, the followingscreening methods may be exemplified.

1) Screening Method Which Uses Phosphorylation of Akt2

It is known that the kinase activity of Akt2 is accelerated when the473rd serine (Ser 473) or the 308th threonine (Thr 308) in the moleculeis phosphorylated (Biochem. J., 1998, 335 (1-13)). Making use of this,the presence or absence of Akt2 activity may be detected by detectingphosphorylated condition of the Ser 473 or Thr 308 of Akt2 by a westernblotting which uses an antibody capable of specifically reacting withthese phosphorylated residues (e.g., anti-phosphoSer antibody or thelike).

A testing cell expressing a part or entire portion of the polypeptidefor screening of the present invention is untreated or treated with asubstance to be tested. As the testing cells, cells which respond toinsulin are desirable, and more illustratively, fat cells, hepatocyte orskeletal muscle-derived cells are desirable. The cell untreated ortreated with a substance to be tested is lysed, and using this as asample, phosphorylation of Akt2, namely the presence or absence the Akt2activity, may be detected making use of western blotting, spot westernblotting or the like method which uses the anti-phosphoSer antibody.Preferably, this may be detected by the method of Example 7. In thisdetection system, a substance used for treating a sample in whichacceleration of the phosphorylation of Akt2 (namely activation of Akt2)was observed in comparison with a sample untreated with the substance tobe tested may be selected as a substance which inhibits binding of thepolypeptide for screening of the present invention with Akt2, and basedon this, an insulin resistance improving agent and/or carbohydratemetabolism improving agent, namely a substance having diabetesmellitus-treating effect, may be screened. As such a substance, it isdesirable to select a substance which shows an ED50 value of the Akt2phosphorylation acceleration action of 10 μM or less, preferably 1 μM orless, more preferably 0.1 μM or less, in said screening method.

2) Screening Method Which Uses In Vitro Kinase Method

The Akt2 activity can also be detected by an in vitro kinase assaymethod in which uptake of radioactive phosphoric acid based on asubstrate is measured when a histone H2B, a GSK-3 fusion protein or thelike is used as the substrate of Akt2 and allowed to react with animmune precipitate of Akt2. Illustratively, a testing cells expressing apart or entire portion of the polypeptide for screenings of the presentinvention are untreated or treated with a substance to be tested. As thetesting cells, cells which responds to insulin are desirable, and moreillustratively, fat cells, hepatocyte or a skeletal muscle-derived cellsare desirable. Activated Akt2 protein may be concentrated from theaforementioned cells by immunoprecipitation using an anti-Akt2 antibody.By mixing a substrate of Akt2, such as GST-crosstide (GST fusion proteinof GSK3-beta sequence as a physiological substrate of Akt), withconcentrated Akt2 protein, kinase activity of Akt2 may be measured anddetermined using phosphorylation of the substrate as the index.Preferably, this may be measured by the method described in Example 7.It is possible to use the kinase measurements as screening methods of alarge number of compound, by making use of the total kinase assaymethods (Waga et al., J. immnunol. Methods, 190, pp. 71-77, 1996). Inthese measuring systems, substances used for treating samples in whichacceleration of the kinase activity of Akt were observed in comparisonwith samples untreated with the substances to be tested may be selectedas substances which inhibits binding of the polypeptide for screening ofthe present invention with Akt2, and based on this, insulin resistanceimproving agents and/or carbohydrate metabolism improving agents, namelysubstances having diabetes mellitus-treating effects, may be screened.As such substances, it is desirable to select a substance which shows anED50 value of the Akt2 kinase acceleration action of 10 μM or less,preferably 1 μM or less , more preferably 0.1 μM or less, in saidscreening methods.

3) Screening Method Which Uses Binding of the Polypeptide for Screeningof the Present Invention with Akt2

Since the polypeptide for screening of the present invention negativelycontrols the insulin signal via its binding to Akt2, the followingscreening method which uses binding of the polypeptide for screening ofthe present invention with Akt2 as the index may be exemplified.Illustratively, testing cells expressing a part or entire portion of thepolypeptide for screenings of the present invention, or a part or entireportion of the polypeptide for screening of the present invention whichis fused with a tag (e.g., GST, Flag, His or the like), is untreated ortreated with a substance to be tested. As the testing cells, cells whichrespond to insulin is desirable, and more illustratively, fat cells,hepatocyte or skeletal muscle-derived cells are desirable. The Akt2protein and a protein binding thereto may be concentrated from theaforementioned cells by immunoprecipitation using an anti-Akt2 antibody.In this concentration step, it is desirable that the same substance tobe tested used in the aforementioned treatment of cells are contained inthe reaction solution. A substance to be tested which inhibits bindingof the polypeptide for screening of the present invention with Akt2 maybe selected by separating the thus obtained concentrated solution ofAkt2 and its binding protein by polyacrylamide gel electrophoresis usinga conventionally known method and measuring the amount of thepolypeptide for screening of the present invention by western blottingusing an antibody. As such a substance, it is desirable to select asubstance which shows an IC50 value, of the action to inhibit binding ofthe polypeptide of the present invention with Akt2, of 10 μM or less,preferably 1 μM or less, more preferably 0.1 μM or less, in theaforementioned screening method. Regarding the antibody to be used inthis case, an antibody specific for the polypeptide for screening of thepresent invention or for the polypeptide for screening of the presentinvention prepared based on its partial sequence (e.g., anti-AKBP2antibody), or an antibody which recognizes the aforementioned tag, maybe used.

In the above screening methods of 1) to 3), the testing cells may beused by un-stimulating or stimulating with insulin, but preferably, thetesting cells may be used by carrying out insulin stimulation.

In addition, substances to be tested which inhibits binding of thepolypeptide for screening of the present invention with Akt2 can also beselected by combining a western blotting similar to the aforementionedone with an in vitro pull down method (H. Matsushime et al., JikkenKogaku (Experimental Engineering), Vol. 13, No. 6, p. 528, 1994, ),using Akt2 protein purified by attaching a tag (e.g., GST or the like)from an extract of cells in which the polypeptide for screening of thepresent invention is expressed, or a mixed solution of proteins preparedby carrying out in vitro transcription and translation, to which asubstance to be tested is added or not added. Preferably, substances tobe tested which inhibits binding of Akt2 with the polypeptide forscreening of the present invention can also be selected using a mixedsolution of proteins prepared by directly carrying out in vitrotranscription and translation of a protein consisting of the polypeptideof the present invention (e.g., AKBP2 protein) as shown in Example 6,from a plasmid which expresses the polypeptide for screening of thepresent invention (e.g., the AKBP2 expression plasmid prepared inExample 1(5)) using an in vitro translation kit (e.g., TNT Kit(Promega)). Each of these methods renders possible screening of a largenumber of substances to be tested by not carrying out polyacrylamide gelelectrophoresis, but carrying out conventionally known spot westernblotting. Also, it is possible to carry out a screening for selecting asubstance to be tested capable of inhibiting binding of Akt2 with thepolypeptide for screening of the present invention, in accordance withconventionally known ELISA methods, which comprises adding a substanceto be tested to a lysate of cells in which the polypeptide for screeningof the present invention expressed by fusing a tag similar to theaforementioned one and Akt2 are simultaneously expressed. In addition,it is possible to select a substance to be tested which inhibits bindingof Akt2 with the polypeptide for screening of the present invention byscreening it from the great majority of population through the detectionof the existing CAT or luciferase activity, making use of theconventionally known two hybrid system for mammal cells (Clontech), andarranging Akt2 fused with the DNA binding region of GAL 4 as the bait,and the polypeptide for screening of the present invention fused withthe transcription accelerating region of VP 16 to the pray side.

<Method for Producing a Pharmaceutical Composition for InsulinResistance Improvement and/or Carbohydrate Metabolism Improvement>

Also included in the present invention is a method for producing apharmaceutical composition for insulin resistance improvement,characterized in that it comprises a step of carrying out screeningusing the polypeptide for screening of the present invention, and a stepof preparing a pharmaceutical preparation.

The pharmaceutical composition which contains a substance obtained bythe screening method of the present invention as the active ingredientmay be prepared using carriers, fillers and/or other additive agentsgenerally used in making pharmaceutical preparations, in response to thetype of the aforementioned active ingredient.

As its administration, oral administration by tablets, pills, capsules,granules, fine subtilaes, powders, solutions for oral use or the like,or parenteral administration by injections for intravenous injection,intramuscular injection, intraarticular injection or the like,suppositories, percutaneous administration preparations, transmucosaladministration preparations or the like may be cited. Particularly,parenteral injection is desirable in the case of peptides which aredigested in the stomach, intravenous injection or the like.

In the solid composition for use in the oral administration, one or moreactive substances may be mixed with at least one inert diluent such aslactose, mannitol, glucose, microcrystalline cellulose,hydroxypropylcellulose, starch, polyvinyl pyrrolidone, aluminummagnesium silicate or the like. In the usual way, the aforementionedcomposition can contain other additives than the inert diluent, such asa lubricant, a disintegrating agent, a stabilizing agent, a solubilizingor solubilization assisting agent or the like. As occasion demands,tablets or pills may be coated with a sugar coating or with a film of agastric or enteric substance or the like.

The liquid composition for oral administration can contain, for example,emulsions, solutions, suspensions, syrups, elixirs or the like and cancontain a generally used inert diluent such as purified water or ethylalcohol. The aforementioned composition can contain additive agentsother than the inert diluent, such as a moistening agent, a suspendingagent, a sweetener, an aromatic, or an antiseptic.

The injections for parenteral administration can include aseptic aqueousor non-aqueous solutions, suspensions or emulsions. The aqueoussolutions or suspensions can include, for example, distilled water forinjection, physiological saline or the like as the diluent. As thediluent for use in the non-aqueous solutions or suspensions, propyleneglycol, polyethylene glycol, plant oil (e.g., olive oil), alcohols(e.g., ethanol), polysorbate 80 or the like can for example be included.The aforementioned composition can further contain, a moistening agent,an emulsifying agent, a dispersing agent, a stabilizing agent, asolubilizing or solubilization assisting agent, an antiseptic or thelike. The aforementioned composition may be sterilized, for example, byfiltration through a bacteria retaining filter, blending of a germicideor irradiation. Alternatively, it may be used by producing a sterilesolid composition and dissolving it in sterile water or other sterilesolvent for injection prior to its use.

The dose may be optionally decided by taking into consideration strengthof activity of the active ingredient, namely the substance obtained bythe screening method of the present invention, symptoms, age, sex andthe like of each patient to be treated.

For example, in the case of oral administration, the dose is generallyapproximately from 0.1 to 100 mg, preferably from 0.1 to 50 mg, per dayper adult (as 60 kg body weight). In the case of parenteraladministration in the form of injections, it is from 0.01 to 50 mg,preferably from 0.01 to 10 mg, per day.

EXAMPLES

The present invention is described in the following based on examples,but the invention is not restricted by said examples. In thisconnection, unless otherwise noted, these may be carried out inaccordance with conventionally known methods (“Molecular Cloning,Sambrook et al., Cold Spring Harbor Laboratory Press, 1989” and thelike). Also, when commercially available reagents and kits are used,these may be carried out in accordance with the instructions attachedthereto.

Example 1 Cloning of Mouse AKBP2 Gene and Construction of ExpressionVector

(1) Cloning of Akt2 Gene

Using the oligonucleotides represented by SEQ ID NO:5 and SEQ ID NO:6,designed with reference to the cDNA sequence described in the accessionnumber M95936 of a gene data base GenBank, as primers, and a humanskeletal muscle cDNA (Marathon-Ready™ cDNA; Clontech) as the template,PCR was carried out using a DNA polymerase (Pyrobest DNA Polymerase(Takara Shuzo)) under a condition of thermal denaturation at 95° C. for3 minutes, 40 repetition of a cycle consisting of 98° C. for 10 seconds,60° C. for 30 seconds and 74° C. for 1 minute and 30 seconds, andfurther heating at 74° C. for 7 minutes. Human Akt2 cDNA was cloned byinserting the resulting DNA fragment of about 1.5 kbp into the EcoRVrecognition site of a plasmid pZErO™-2.1 (Invitrogen). Nucleotidesequence of the Akt2 cDNA cloned on the vector was determined by asequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNAsequencer, Applied Biosystems), using the aforementionedoligonucleotides represented by SEQ ID NO:5 and SEQ ID NO:6, therebyconfirming that its sequence coincided with the reported sequence.

(2) Preparation of Expression Plasmid for Yeast Two Hybrid

In order to insert the human Akt2 cDNA into an expression vector foryeast two hybrid, pDBtrp (Invitrogen), primers represented by SEQ IDNO:7 and SEQ ID NO:8 were designed by adding a region homologous with 40nucleotides in front and in the rear of the pDBtrp vector multi-cloningsite to the 5′-side and 3′-side of the human Akt2 gene sequence. PCR wascarried out using the Akt2 plasmid cloned in the above as the templateand using a DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo), byheating at 98° C. (1 minute) and then repeating 35 times of a cycleconsisting of 98° C. (5 seconds), 55° C. (30 seconds) and 72° C. (5minutes) The DNA fragment obtained as the result has the complete coderegion of the human Akt2 gene.

The vector pDBtrp made into a linear chain by digesting with restrictionenzymes SalI and NcoI and the PCR fragment containing Akt2 cDNA obtainedin the above were simultaneously added to yeast strain MaV203 for twohybrid (Invitrogen) which was then transformed by a lithium acetatemethod (Guthrie C. and Fink R., Guide to Yeast Genetics and MolecularBiology, Academic, San Diego, 1991). As a result, homologousrecombination occurred in the yeast cell, and a plasmid in which theAkt2 cDNA was inserted into the multi-cloning site of pDBtrp (to bereferred to as pDB-Akt2 hereinafter) was formed. Yeast cell having thepDB-Akt2 plasmid were selected by culturing on a solid synthetic minimalmedium (DISCO,20% agarose) from which tryptophan as a selection markerof the plasmid had been deleted, the yeast cells were treated withzymolyase (Seikagaku Kogyo) at 37° C. for 30 minutes, and then theplasmids were isolated and purified by the alkali method, anddetermination of their nucleotide sequences was carried out using asequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNAsequencer, Applied Biosystems) to select those in which the Akt2 cDNAwas inserted together with the code region and translation frame of GAL4 DNA binding region of pDBtrp.

(3) Preparation of Mouse Fat Tissue Derived cDNA Library

By purchasing C57BL/6J male mice of 12 weeks of age and C57BL/KsJ-+m/+mmale mice of 13 weeks of age from CLEA Japan, Poly(A)+RNA was preparedfrom epididymis fat in accordance with the mRNA preparation methoddescribed in Experimental Medicine Supplement, Bio-manual Series 2, GeneLibrary Preparation Method (written in Japanese, edited by H. Nojima;published by Yohdosha on Feb. 20, 1994). Using ZAP-cDNA Synthesis Kitmanufactured by Stratagene and in accordance with the protocol attachedthereto, first-strand synthesis and second-strand synthesis were carriedout using 5 μg of RNA, and the double-stranded cDNA was smooth-ended,ligated with the EcoRI adapter attached to the kit and then digestedwith restriction enzymes EcoRI and XhoI. Size fractionation was carriedout using a spin column (CHROMA SPIN-400; Clontech), and shorterfragments were removed. A 100 μg portion of a vector pACT2 (Clontech)was digested with the restriction enzyme XhoI, treated with an alkalinephosphatase (Bacterial Alkaline Phosphatase; Takara Shuzo), and thendigested with the restriction enzyme EcoRI and applied to a spin column(CHROMA SPIN-1000; Clontech). In accordance with the cDNA librarypreparation method described in Experimental Medicine Supplement,Bio-manual Series 2, Gene Library Preparation Method (edited by H.Nojima; published by Yohdosha on Feb. 20, 1994), the vector and cDNAwere ligated, and the sample after ligation was treated with a filtercup (UFCP3TK50) manufactured by Millipore. Using the Escherichia colifor electroporation manufactured by GIBCO BRL (ElectroMAXX DH10B™Cells), transformation was carried out by the electroporation method,and cultured overnight on a shaker using 1,000 ml of a culture medium.After confirming that 10⁶ or more of independent colonies are present inthe culture medium, plasmids were purified using a plasmid purificationkit (Qiagen Plasmid Kit; Qiagen) and in accordance with the protocolattached to the kit.

(4) Yeast Two Hybrid Screening

The aforementioned yeast strain MaV203 for two hybrid transformed bypDB-Akt2 was suspended in 400 ml of YPD liquid medium (DIFCO), culturedat 30° C. for about 6 hours until absorbance at a wave length of 590nanometer became from 0.1 to 0.4, and then made into competent cells bythe lithium acetate method, and the final amount was suspended in 1.0 mlof 0.1 M lithium-tris buffer. The cells were transformed with 20 μg ofthe mouse fat tissue derived cDNA library prepared in the aforementioned(3), and the cells were selected by culturing on a solid syntheticminimal medium (DISCO, 20% agarose) from which tryptophan and leucine asrespective selection markers of pDB-Akt2 and the library had beendeleted, thereby obtaining a transformant into which both plasmids wereintroduced. At the same time, in order to select a cell having anactivated reporter gene HIS3 which is expressed when a fusion protein ofthe GAL4 DNA binding domain artificially expressed in the two hybridsystem is linked to a fusion protein of the GAL4 transcriptionalactivation domain, the transformed cells were cultured at 30° C. for 5days on the solid minimal medium (20% agarose) from which histidine wasremoved together with tryptophan and leucine and to which 20 mM of 3AT(3-amino-1,2,4-triazole; Sigma) as an inhibitor of the enzyme encoded byHIS3 was added. The 3AT-resistant yeast colonies showing that a proteinwhich binds to Akt2 is expressed under the same condition were obtained.These yeast cells were grown on the YPD solid medium for 24 hours, andthen expression of the lacZ gene which is different from HIS3 but is abinding indicator reporter of the two hybrid system was examined usingβ-galactosidase activity as the index. Regarding the β-galactosidaseactivity, yeast cells on the medium were transferred on a nitrocellulosefilm, frozen in liquid nitrogen and then thawed at room temperature, andthe filter was put on a filter paper soaked with 0.4% X-GAL (Sigma)solution and allowed to stand at 37° C. for 24 hours to measure changeof color to blue caused by β-galactosidase. By selecting colonies inwhich contents of cells transferred on the filter changed from white toblue, yeast cells expressing a protein which binds to Akt2 werespecified, and library derived plasmids were extracted from the cells inaccordance with the method of Yeast Protocols Handbook of Clontech.Nucleotide sequences of the gene fragments contained therein weredetermined by a sequencing kit (Applied Biosystems) and a sequencer (ABI3700 DNA sequencer, Applied Biosystems) using the nucleotide sequencerepresented by SEQ ID NO:9 (a sequence which binds to the GAL4 ADregion; derived from GenBank accession number U29899 Cloning vectorpACT2) as the primer, and it was confirmed as a result that thenucleotide sequence represented by SEQ ID NO:1 was contained therein.

(5) Determination of Initiation Codon of Mouse AKBP2 Gene

As a result of the aforementioned (4), a library derived plasmid havinga gene fragment containing the nucleotide sequence represented by SEQ IDNO:1 was obtained. Accordingly, in order to determine initiation codonof the gene contained in said fragment, a primer of the nucleotidesequence represented by SEQ ID NO:10 which corresponds to acomplementary chain of a nucleotide sequence of from the 1034th positionto the 1011th position of the nucleotide sequence represented by SEQ IDNO:1 was synthesized (Proligo), and an attempt was made to amplifycomplete length cDNA derived from an expression product of said genefrom the aforementioned fat tissue-derived cDNA library by PCR usingsaid primer and the aforementioned primer of the nucleotide sequencerepresented by SEQ ID NO:9. The PCR was carried out using a DNApolymerase (TAKARA LA Taq; Takara Shuzo) and by heating at 94° C. (3minutes) and then repeating 35 times of a cycle consisting of 94° C. (30seconds), 58° C. (1.5 minutes) and 72° C. (4 minutes). The DNA fragmentsin the reaction solution were cloned into an expression vector(pcDNA3.1/V5-His-TOPO; Invitrogen) using TOPO TA Cloning System(Invitrogen). Nucleotide sequences of the inserted DNA fragments in thethus obtained plasmids were determined using a primer (TOPO TA CloningKit; Invitrogen; SEQ ID NO:11) which binds to the T7 promoter region onthe vector, a sequencing kit (Applied Biosystems) and a sequencer (ABI3700 DNA sequencer, Applied Biosystems). As a result, plasmidscontaining cDNA molecules of various lengths having the sequence of saidgene were obtained, but chain lengths of the longest cDNA molecules werealmost the same as that of the transcription product derived cDNAobtained in the aforementioned (4). Since several trials showed the sameresult, it was found that the chain length of sequence of thetranscription product of said gene almost coincide with those of thecDNA obtained in (4). Based on this, it was found that the first ATG ofthe nucleotide sequence represented by SEQ ID NO:1 is the initiationcodon of said gene, so that the open reading frame of said generepresented by SEQ ID NO:1 was confirmed. This gene was named mouseAKBP2 gene.

(6) Preparation of Mouse AKBP2 Expression Vector

As a result of the aforementioned (4), a library-derived plasmid havinga gene fragment containing complete length of the nucleotide sequencerepresented by SEQ ID NO:1 was obtained, and the presence of a factorwhich binds to Akt2 was indicated. Also, its open reading frame wasconfirmed in the aforementioned (5). Accordingly, the primersrepresented by SEQ ID NO:12 and SEQ ID NO:13 were synthesized (Proligo)in accordance with the nucleotide sequence information shown in SEQ IDNO:1, and an AKBP2 cDNA coding for the net AKBP2 protein was amplifiedby PCR using said primers and the plasmid obtained in the aforementioned(4) as the template. These two kinds of DNA primers respectively havenucleotide sequences homologous with partial sequences of the 5′-sideand 3′-side of the mouse AKBP2 gene represented by SEQ ID NO:1. PCR wascarried out using a DNA polymerase (Pyrobest DNA Polymerase; TakaraShuzo), by heating at 98° C. (1 minute) and then repeating 35 times of acycle consisting of 98° C. (5 seconds), 55 ° C. (30 seconds) and 72° C.(5 minutes) As a result of separating the PCR product by an agarose gelelectrophoresis, it was confirmed that a DNA fragment of about 1.7 kbpwas amplified. Accordingly, this DNA fragment in the reaction solutionwas subcloned into an expression vector (pcDNA3.1/V5-His-TOPO;Invitrogen) using TOPO TA Cloning System (Invitrogen). The primerrepresented by SEQ ID NO:13 used in this case was designed in such amanner that the stop codon sequence of AKBP2 was removed so that avector-derived VS epitope (derived from V protein of paramyxovirus SV 5,Southern J. A., J. Gen. Virol., 72, 1551-1557, 1991) and His 6 tag(Lindner P., BioTechniques, 22, 140-149, 1997) were continued with thesame frame of the triplet of mouse AKBP2 gene at the 3′-side aftercloning. Nucleotide sequence of the inserted DNA fragment in the thusobtained plasmid was determined using a primer (TOPO TA Cloning kit;Invitrogen; SEQ ID NO:11) which binds to the T7 promoter region on thevector, a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700DNA sequencer, Applied Biosystems). As a result, it was confirmed thatthe 1719 base pair AKBP2 cDNA represented by SEQ ID NO:1 coding for thenet AKBP2 protein was inserted into the aforementioned expression vectorpcDNA3.1/V5-His-TOPO, as a DNA from which the 3′-side stop codon of theDNA sequence was removed. This expression plasmid is referred to aspcDNA-AKBP2 hereinafter.

Example 2 Preparation of Cultured Cell Which Expresses AKBP2 Protein

(1) Preparation of AKBP2 Expression Cell

The aforementioned expression plasmid pcDNA-AKBP2 prepared in Example1(5) or an empty vector (pcDNA3.1/V5-His-TOPO) was introduced into the293 cell (Cell Bank). The 293 cell was cultured in a culture dish of 6well culture plate (well diameter 35 mm) until it became a state of 70%confluent, by adding 2 ml of a minimal essential medium DMEM (Gibco)containing 10% fetal bovine serum (Sigma) to each well. The pcDNA-AKBP2(3.0 μg/well) was transiently introduced into this cell by the calciumphosphate-method (Graham et al., Virology, 52, 456, 1973; N. Arai, GeneTransfer and Expression/Analytical Method (written in Japanese), pp.13-15, 1994). After 30 hours of culturing, the medium was removed andthe cells were washed with phosphate buffered saline (to be referred toas PBS hereinafter), and then the cells were lysed by adding 0.1 ml perwell of a cell lysis solution (100 mM potassium phosphate (pH 7.8), 0.2%Triton X-100).

(2) Detection of AKBP2 Protein

A 10 μl portion of 2× SDS sample buffer (125 mM Tris-HCl (pH 6.8), 3%sodium lauryl sulfate, 20% glycerol, 0.14 M β-mercaptoethanol, 0.02%Bromophenol Blue) was added to 10 μl of the aforementioned lysate ofAKBP2 expression cell of Example 2(1), and this was treated at 100° C.for 2 minutes and then subjected to 10% SDS polyacrylamide gelelectrophoresis to separate proteins contained in the sample. Proteinsin the polyacrylamide gel were transferred on a nitrocellulose membraneusing a semi-dry type blotting device (Bio-Rad), and then detection ofthe AKBP2 protein on said nitrocellulose membrane was carried out bywestern blotting in accordance with the usual way. A monoclonal antibody(Invitrogen) which recognizes V5 epitope fused to the C-terminus ofAKBP2 was used as the primary antibody, and mouse IgG-HRP fusionantibody (Bio-Rad) was used as the secondary antibody. As a result, asshown in FIG. 1, it was confirmed that a protein of about 70 kDa whichcorresponds to an AKBP2-V5-His 6 fusion protein consisting of 618 aminoacids containing the C-terminal side tag consisting of 45 amino acid isdetected dependently on the gene transfer of the expression vectorpcDNA-AKBP2. Based on this, it was revealed that complete length regionof the aforementioned mouse AKBP2 gene cloned is certainly expressed andcan form stable structure as the protein in the cultured cell.

Example 3 Measurement of AKBP2 Expression Level in Normal Mice, High FatDiet-loaded Normal Mice and Diabetes Mellitus Model Mice

Based on the aforementioned information, it was found that the mouseAKBP2 protein of the present invention binds to Akt2, and is expressedin the insulin-responding tissues including both adipose and muscle.Since Akt2 protein is a factor which acts upon the insulin signal firstpathway, it was considered that action of the AKBP2 of the presentinvention is related to the insulin resistance. Accordingly, measurementof messenger RNA (mRNA) expression level of the AKBP2 gene in muscle andfat was carried out using a type 2 diabetes mellitus model miceKKA^(y)/Ta (Iwatsuka et al., Endocrinol. Japon.: 17, 23-35, 1970,Taketomi et al., Horm. Metab. Res., 7, 242-246, 1975) and a healthy miceC57BL/6J fed with a normal feed or a high fat diet.

Regarding the gene expression level, expression level of the mouse AKBP2gene of the present invention was measured and corrected by thesimultaneously measured expression level of glyceraldehyde 3-phosphatedehydrogenase (G3PDH) gene. As the measuring system, PRISM™ 7700Sequence Detection System and SYBR Green PCR Master Mix (AppliedBiosystems) were used. In this measuring system, expressed amount of thegene of interest is determined by detecting and monitoring fluorescencelevel of the SYBR Green I pigment incorporated by double-stranded DNAamplified by PCR in a real time manner.

Illustratively, the measurement was carried out by the followingprocedures.

(1) Preparation of Total RNA

C57BL/6J male mice of 14 weeks of age loaded with a usual feed or a highfat diet, and C57BL/6J male mice and KKA^(y)/Ta mice of 15 weeks of age(all from CLEA Japan) were used. The high fat diet loading was carriedout for 9 weeks from 5 weeks of age to 14 weeks of age. Composition ofthe high fat diet is as follows: casein 29.8%, sucrose 15.8%, vitaminmix 1.3%, mineral mix 8.8%, cellulose powder 5.0%, methionine 0.5%,safflower oil 28.9%, water 10%. On the other hand, CE-2 (CLEA Japan) wasused as the normal feed. Muscle and fat of each of the aforementionedmice were extracted, and total RNA was prepared using a reagent for RNAextraction (Isogen; Nippon Gene) and in accordance with itsinstructions. Each total RNA thus prepared was then treated usingdeoxyribonuclease (Nippon Gene), subjected to phenol/chloroformtreatment and ethanol precipitation, dissolved in sterile water andstored at −20° C.

(2) Synthesis of Single-stranded cDNA

Reverse transcription of total RNA into single-stranded cDNA was carriedout in a system of 20 μl using 1 μg of RNA (fat), 1 μg of RNA (muscle ofa mouse of 14 weeks of age) or 0.25 μg of RNA (muscle of a mouse of 15weeks of age) prepared in (1), and using a kit for reverse transcriptionreaction (Advantage™ RT-for-PCR Kit; Clontech). After the reversetranscription, this was mixed with 180 μl of sterile water and stored at−20° C.

(3) Preparation of PCR Primers

Four oligonucleotides (SEQ ID NO:14 to SEQ ID NO:17) were designed asthe PCR primers described in the following item (4). They were used as acombination of SEQ ID NO:14 with SEQ ID NO:15 for the mouse AKBP2 gene,and a combination of SEQ ID NO:16 with SEQ ID NO:17 for the G3PDH gene.

(4) Measurement of Gene Expression Quantity

Real time measurement of PCR amplification by PRISM™ 7700 SequenceDetection System was carried out in a system of 25 μl in accordance withthe instructions. In each system, 5 μl of single-stranded cDNA, 12.5 μlof 2×SYBR Green reagent and 7.5 pmol of each primer were used. In thiscase, the single-stranded cDNA preserved in (2) was used by diluting 30times regarding the G3PDH, or by diluting 10 times regarding the mouseAKBP2. Instead of the single-stranded cDNA, 0.1 μg/μl of a mouse genomicDNA (Clontech) was appropriately diluted and a 5 μl portion thereof wasused for the preparation of calibration curve. PCR was carried out byheating at 50° C. for 10 minutes, subsequently heating at 95° C. for 10minutes, and then repeating 45 cycles of a process consisting of 2 stepsof 95° C. for 15 seconds and 60° C. for 60 seconds.

Expressed amount of the mouse AKBP2 gene in each sample was corrected bythe expressed amount of G3PDH gene based on the following formula.[Corrected amount of AKBP2 expression]=[expressed amount of AKBP2 (rawdata)]/[expressed amount of G3PDH (raw data)]

FIG. 2 shows relative amounts in which the expressed amount in C57BL/6Jmouse of usual feed was defined as 1 in comparing expressed amounts infat, and the expressed amount in C57BL/6J also as 1 in comparingexpressed amounts in muscle tissue.

As shown in FIG. 2, it was confirmed that expression of the mouse AKBP2gene of the present invention is markedly increased in the fat andmuscle at the time of high fat diet loading, or in the fat and muscle ofthe diabetes mellitus model mouse. Accordingly, it is considered thatthe mouse AKBP2 of the present invention induces insulin resistance bythe acceleration of expression quantity in fat and muscle. Based on theabove, it may be concluded that concern of the mouse AKBP2 of thepresent invention in the insulin resistance is large.

In addition, it was revealed from the results of this Example thatdiagnosis of diabetes mellitus morbid state may be made by measuringexpression quantity of mouse AKBP2.

Example 4 Cloning of Human AKBP2 Gene, and Its Expression DistributionAnalysis in Various Tissues

An attempt was made on the amplification of AKBP2 human orthologue genecomplete length cDNA by the same PCR method shown in the aforementionedExample 1(5), using a human fat-derived cDNA library (Clontech) as thetemplate and a pair of primers represented by SEQ ID NO:18 and SEQ IDNO:19. When nucleotide sequence of a DNA fragment of about 1.8 kbpobtained as a result thereof was determined in accordance with the samemethod shown in Example 1, it was confirmed that it contains completelength cDNA of the gene represented by SEQ ID NO:3. Said gene cDNA is anovel gene which encodes the polypeptide represented by SEQ ID NO:4.Said gene is a human orthologue gene of AKBP2 in which it has a homologyof 76.8% with the. mouse AKBP2 gene represented by SEQ ID NO:1, and theencoded polypeptide has a homology of 71.3% with the mouse AKBP2 proteinrepresented by SEQ ID NO:2, respectively.

Accordingly, an attempt was subsequently made to amplify a cDNA fragmentof about 800 bases of the 3′-side of the human AKBP2 gene from cDNAsamples derived from various human tissues, by PCR using the primerrepresented by SEQ ID NO:20 newly designed based on the sequence of saidhuman AKBP2 gene and the aforementioned primer represented by SEQ IDNO:19, and the presence or absence of the expression of AKBP2 inrespective tissues was examined. The PCR was carried out by DNApolymerase (Pyrobest DNA polymerase; Takara shuzo) using 2 μg of each ofthe various human tissue cDNA libraries (Clontech) as the template and,after heating at 98° C. (1 minute), repeating 35 cycles each cycleconsisting of 98° C. (5 seconds), 55° C. (30 seconds) and 72° C. (5minutes). When the thus obtained PCR products were separated by anagarose gel electrophoresis, a desired DNA fragment of about 800 basepair considered to be containing a 3′-terminal side partial sequence ofthe human AKBP2 gene was amplified from each of the skeletal muscle-,liver- and fat-derived cDNA libraries. When these DNA fragments wereseparated from respective agarose gels, and nucleotide sequences of saidDNA fragments were respectively determined in accordance with the methoddescribed in the aforementioned Example 1(4) using the primerrepresented by SEQ ID NO:20, it was confirmed that they are the3′-terminal side partial sequence of human AKBP2 gene represented by SEQID NO:3. Based on this, it was revealed that expression of the humanAKBP2 gene is specifically controlled in fat, muscle, liver and the likelimited organs which respond to the insulin signal.

As a result of this Example, since the human AKBP2 showed high homologywith mouse AKBP2 and its expression was observed in insulin respondingtissues, it was confirmed that it has the same functions of those of themouse counterpart and therefore is useful for the diagnosis of diabetesmellitus and screening of a diabetes mellitus improving agent.

Example 5 Preparation of Cultured Cells Which Express Human AKBP2Protein

The aforementioned AKBP2 gene complete length cDNA obtained in Example 4was subcloned by the same method shown in the aforementioned Example1(6). Thereafter, it was confirmed that the 1782 base pair human AKBP2cDNA represented by SEQ ID NO:3 coding for the net human AKBP2 proteinwas inserted into the aforementioned expression vectorpcDNA3.1/V5-His-TOPO, as a DNA from which the 3′-side stop codon of theDNA sequence was removed. This expression plasmid is referred to aspcDNA-human AKBP2 hereinafter. By introducing 5.1 μg per well of thisexpression plasmid pcDNA-human AKBP2 by the same method of Example 2(1),expression of human AKBP2 protein was detected in accordance with themethod of Example 2(2). As a result, it was confirmed that a protein ofabout 70 kDa which corresponds to a human AKBP2-V5-His 6 fusion proteinconsisting of 638 amino acids containing the C-terminal side tagconsisting of 45 amino acid is detected dependently on the gene transferof the expression vector pcDNA-human AKBP2, so that it was revealed thatthe complete length region of the aforementioned cloned human AKBP2 geneis certainly expressed in the cultured cells and can form stablestructure as the protein.

Example 6 Inspection of Interaction Between Human AKBP2 and Akt2

(1) Preparation of GST Fusion Akt2 Expression Plasmid

In order to insert human Akt2 cDNA into a GST fusion expression vectorpGEX-3X (Amersham Bioscience), the human Akt2 cDNA obtained in Example1(1) was digested with restriction enzymes HindIII and EcoRI, and thevector PGEX-3X with restriction enzymes BamHI and EcoRI, respectively,thereby making them into linear chains. In addition, in order to use thefragments represented by SEQ ID NO:21 and SEQ ID NO:22 as fragments fortheir ligation, they were separately treated at 60° C. for 30 minutes asa pretreatment and then mixed and allowed to stand at room temperaturefor 2 hours. A mixture of these treated human Akt2 cDNA fragment, vectorpGEX-3X and fragment for ligation was mixed with a DNA ligase solution(DNA ligation kit II; Takara Shuzo) and treated at 16° C. for 3 hours,thereby preparing a plasmid in which Akt2 cDNA was inserted into themulti-cloning site of pGEX-3X (to be referred to as pGEX-Akt2hereinafter). By carrying out determination of the nucleotide sequenceusing the oligonucleotide represented by SEQ ID NO:23 as a primer andusing a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700DNA sequencer, Applied Biosystems), those in which the code region ofAkt2 cDNA and the GST tag translation frame of pGEX vector were insertedin union were selected.

(2) Purification of GST Fusion Akt2 Protein

Using the plasmid pGEX-Akt2 obtained in the aforementioned (1),Escherichia coli BL21 was transformed by the heat shock method andcultured overnight on a shaker using 2.4 ml of a culture medium, totalvolume thereof was inoculated into 400 ml of a culture medium andcultured at 37° C. for 3 hours on a shaker, and then IPTG (SIGMA) wasadded thereto to a final concentration of 2.5 mM and the shakingculturing was further continued for 3 hours to induce expression of theGST fusion Akt2 protein (to be referred to as GST-Akt2 hereinafter). Byrecovering the cells, GST-Akt2 was purified on glutathione Sepharosebeads (Glutathione Sepharose 4B; Amersham Pharmacia) in accordance witha conventionally known method (H. Matsushime et al., Jikken Kogaku(Experimental Engineering), Vol. 113, No. 6, p. 528, 1994). As acontrol, a protein of GST moiety alone (to be referred to as GST proteinhereinafter) was expression-induced and purified from the Escherichiacoli BL21 transformed with pGEX-3X, in the same manner as in the above.By carrying out separation by SDS polyacrylamide gel electrophoresis andCoomassie Brilliant Blue staining in accordance with conventionallyknown methods, it was confirmed that the proteins having expectedmolecular weights (GST-Akt2; 79 kDa, GST protein; 26 kDa) were purified.

(3) Verification of Biochemical Binding of Akt2 Protein with Human AKBP2Protein

Using the GST fusion Akt2 protein (to be referred to as GST-Akt2hereinafter) prepared in the aforementioned (2), the presence or absenceof direct interaction between human AKBP2 protein and Akt2 protein wasverified by the GST-pull down method (H. Matsushime et al., JikkenKogaku, Vol. 113, No. 6, p. 528, 1994). Firstly, using 0.5 μg of thepcDNA-human AKBP2 prepared in the aforementioned Example 5 as thetemplate, and using 40 μl of a TNT kit (TNTR Quick CoupledTranscription/Translation System; Promega) and 1.3 MBq of a radioisotope(redivue Pro-mix L-[³⁵S]; Amersham), radioisotope-labeled human AKBP2protein was prepared by in vitro transcription and translation inaccordance with the attached protocols. A 15 μl portion of this humanAKBP2 protein preparation solution was mixed with 1 μg of the GSTprotein or GST-Akt2 purified on glutathione beads in the aforementioned(2) and shaken at 4° C. for 1 hour after adding 0.3 ml of Buffer A (50mM Tris-HCl (pH 7.5), 10% glycerol, 120 mM NaCl, 1 mM EDTA, 0.1 mM EGTA,0.5 mM PMSF, 0.5% NP-40). Thereafter, the protein which binds to GSTprotein or GST-Akt2 on beads was co-precipitated by centrifugation. Thiswas suspended in 0.5 ml of a buffer solution in which the NaClconcentration of the aforementioned Buffer A was changed to 100 mM, andagain co-precipitated by centrifugation. After repeating this operation4 times, proteins in the precipitate were separated by SDSpolyacrylamide gel electrophoresis in accordance with a conventionallyknown method, and the human AKBP2 protein was detected byautoradiography. As a result, a band which is not detected when the GSTprotein is mixed was detected when GST-Akt2 was mixed. Based on this, itwas revealed that the human AKBP2 as one of the polypeptides of thepresent invention interacts with Akt2 protein similar to the case of themouse AKBP2 of the present invention, thus proving that these human andmouse AKBP2 molecules are counterparts which carry out the same functionin both animal species. Accordingly, it was found that the human AKBP2of the present invention is concerned in the induction of insulinresistance via its interaction with Akt2 protein similar to the case ofthe mouse AKBP2 of the present invention.

Example 7 Influence of Mouse AKBP2 Over-expression in NIH3T3 L1 Fat CellUpon Akt2 Kinase Activity

From the results of the aforementioned yeast two hybrid and biochemicalbinding analyses, it was shown that Akt2 and AKBP2 interact with eachother. Accordingly, influence of AKBP2 upon the enzyme (kinase) activityof Akt2 was examined by an in vitro kinase assay using a cultured cellNIH3T3 L1.

(1) Preparation of Substrate GST-crosstide for In Vitro Kinase Assay

The synthetic oligo DNA molecules represented by SEQ ID NO:24 and SEQ IDNO:25 coding for the phosphorylation region of GSK3p which is aphysiological substrate of Akt2 were mixed and integrated into the EcoRIXhoI site of the pGEX-6P-1 vector. This was used as GST-crosstide. Usingthe plasmid GST-crosstide, Escherichia coli BL21 was transformed by theheat shock method and cultured overnight on a shaker using 2.4 ml of aculture medium, total volume thereof was inoculated into 400 ml of aculture medium and cultured at 37° C. for 3 hours on a shaker, and thenIPTG (SIGMA) was added thereto to a final concentration of 2.5 mM andthe shaking culturing was further continued for 3 hours to induceexpression of a GST fusion protein (to be referred to as GST-crosstidehereinafter). By recovering the cells, GST-crosstide was purified onglutathione Sepharose beads (Glutathione Sepharose 4B; AmershamPharmacia) in accordance with a conventionally known method (H.Matsushime et al., Jikken Kogaku (Experimental Engineering), Vol. 113,No. 6, p. 528, 1994). By carrying out separation of these proteins bySDS polyacrylamide gel electrophoresis and their Coomassie BrilliantBlue staining in accordance with conventionally known methods, it wasconfirmed that the GST-crosstide was purified.

(2) Preparation of AKBP2 High Expression Virus Making Use of AdenovirusVector

A gene fragment coding for mouse AKBP2 was cut out from the pcDNA-AKBP2vector using restriction enzymes BamHI and SacIl and inserted into themulti-cloning site (for BGlII and NotI) of an adenovirus vectorpAdTrack-CMV (obtained from Johns Hopkins Cancer Center) using linkeroligo SEQ ID NO:26 and SEQ ID NO:27 which form SacII and NotI digestionfragments, thereby obtaining a vector AKBP2/pAdTrack-CMV.

Thereafter, preparation of a solution of high titer adenovirus capableof expressing AKBP2 was carried out in accordance with a conventionallyknown protocol [“A Practical Guide for using the AdEasy System”(HYPERLINK “http://www.coloncancer.org/adeasy.htm”“http://www.coloncancer.org/adeasy/protocol2.htm”)]. The adenovirus forcontrol was prepared from pAdTrack-CMV.

In this connection, regarding the amount of virus, absorbance at 260 nm(A260) was measured and converted by the following formula.1 A260=1.1×10¹² virus particles=3.3×10¹¹ pfu/ml   [Formula]

(3) Over-expression of Mouse AKBP2 in NIH3T3 L1 Fat Cells andImmunoprecipitation of Akt2

NIH3T3 L1 cells were suspended in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal calf serum (FCS) and inoculated in 8×10⁵cells/well potions into a collagen-coated 6 well plate (Asahi TechnoGlass). On the next day, the medium was exchanged with the DMEM (10%FCS) further supplemented with 10 μg/ml of insulin, 250 nM ofdexamethasone and 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX) to inducedifferentiation of 3T3-L1 cells. Two days thereafter, the medium wasreturned to 0.4 ml of DMEM (10% FCS). Four days thereafter, anadenovirus which expresses AKBP2 was added to the medium at aconcentration of 8×10¹⁰ pfu per well. As a control, an adenovirus whichexpresses GFP alone was used.

After 36 hours of the adenovirus infection, the cells were cultured for16 hours using serum-free DMEM medium, stimulated with 100 nM of insulinfor a predetermined period of time (0, 30 or 60 minutes) and thenimmediately dissolved in 500 μl of a cell lysis solution (50 mM Tris-HClpH 7.5, 1 mM EDTA, 5 mM EGTA, 0.5 mM Na₃VO₄, 0.1% 2-mercaptoethanol, 50mM NaF, 5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1% TritonX-100, 0.1 mM PMSF). After centrifugation at 15,000 rpm for 20 minutes,the supernatant was mixed with an anti-Akt2 antibody (Upstate) andProtein G-Sepharose (Amersham) to effect immunoprecipitation. The immuneprecipitate was washed twice with the cell lysis solution, twice with awashing solution (50 mM Tris-HCl pH 7.5, 0.03% Brij 35, 0.1%2-mercaptoethanol) and then twice with a reaction solution (20 mM MOPSpH 7.2, 10 mM MgCl₂, 25 mM β-glycerophosphate, 5 mM EDTA, 1 mM DTT) andsubjected to the kinase reaction.

(4) In Vitro Kinase Assay

The aforementioned immune precipitate was suspended in 20 μl of thereaction solution. The reaction solution further supplemented with 15 μMof ATP, 10 μCi of [γ³²P]-ATP and 3 μg of GST-crosstide was added theretoand heated at 30° C. for 20 minutes. The reaction was stopped by adding10 μl of 4× SDS sample buffer. After separation by SDS polyacrylamidegel electrophoresis, the radioactivity incorporated into GST-crosstidewas determined by analyzing it by BAS 2000 Bioimaging Analyzer (FujiPhoto Film). As shown in FIG. 3, the kinase activity of Akt2 in theNIH3T3 L1 cell was reduced by over-expression with AKBP2 to 0.82 timesas compared with the control (GFP) under insulin-un-stimulated state. Inthe same manner, when activity increase of Akt2 by the 100 nM insulinstimulation was observed, the GFP-infected cells showed 1.39 times (30minutes) and 1.5 times (60 minutes) increase in the enzyme activity incomparison with the un-stimulated state, while the cells infected withthe AKBP2 virus showed only 1.30 times (30 minutes) and 1.22 times (60minutes) of the stimulation dependent increase in the activity. Also,since phosphorylation of the 473rd serine is important for theactivation of Akt2, the aforementioned Akt2 immuno-precipitated afterthe insulin stimulation was analyzed by western blotting using ananti-phosphorylated serine473 antibody (New England Biolab) to find thatthe phosphorylated state at the time of no stimulation was reduced inthe cells infected with the AKBP2 virus in comparison with the controlvirus-infected cells. In addition, a stimulation-dependent accelerationof phosphorylation was found by 30 minutes of insulin stimulation inboth of the cells infected with the AKBP2 virus and the controlvirus-infected cells, but degree of the phosphorylation acceleration wasweakened in the cells infected with the AKBP2 virus than in the controlvirus-infected cells, so that the results of the in vitro kinase assaywas supported. Based on the above results, it is considered that themouse AKBP2 of the present invention interacts with Akt2 and inducesinsulin resistance by reducing increase of the insulin-dependent enzymeactivity, in addition to the insulin-independent enzyme activity.

INDUSTRIAL APPLICABILITY

The polypeptides and polynucleotides of the present invention which havethe property to bind to Akt2, reduce the kinase activity of Akt2 andincrease the expression level in the diabetes mellitus morbid state areuseful for the diagnosis of diabetes mellitus. In addition, thepolypeptides, polynucleotides, expression vectors and cells of thepresent invention are useful for the screening of a substance whichinhibits binding of the polypeptides of the present invention with Akt2(namely a substance which reinforces function of Akt2). The substanceselected by said screening is useful as a candidate substance for aninsulin resistance improving agent and a diabetes mellitus improvingagent.

Sequence Listing Free Text

An explanation of “Artificial Sequence” is described the numerical entry<223> of the following Sequence Listing. Illustratively, each of thenucleotide sequences represented by SEQ ID NOs:5 to 8 and 10 to 27 inthe Sequence Listing is an artificially synthesized primer sequence. Thenucleotide sequence represented by the sequence of SEQ ID NO:9 is asequence consisting of the bases of from the 5183rd (5′) to the 5162nd(3′) positions of a cloning vector pACT2 (GenBank U29899).

In the foregoing, the invention has been described based on thespecified embodiments, but changes and modifications obvious to thoseskilled in the art are included in the scope of the invention.

1. A polypeptide which comprises the amino acid sequence represented bySEQ ID NO:2 or SEQ ID NO:4, or an amino acid sequence in which from 1 to10 amino acids are deleted, substituted and/or inserted in the aminoacid sequence represented by SEQ ID NO:2 or SEQ ID NO:4, and which bindsto Akt2.
 2. A polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO:2 or SEQ ID NO:4.
 3. A polynucleotide codingfor the polypeptide described in claim 1 or claim
 2. 4. An expressionvector comprising the polynucleotide described in claim
 3. 5. A celltransformed with the expression vector described in claim
 4. 6. A methodfor screening a substance which inhibits binding of a polypeptidedescribed in claim 1 or claim 2 or a polypeptide consisting of an aminoacid sequence having a homology of 90% or more with the amino acidsequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds toAkt2, with Akt2, which comprises allowing (1) the aforementionedpolypeptide or a cell expressing the aforementioned polypeptide, tocontact (2) a substance to be tested, measuring binding of saidpolypeptide with Akt2, and selecting a substance which inhibits theaforementioned binding.
 7. The screening method described in claim 6,wherein the binding inhibiting substance is an insulin resistanceimproving agent and/or a carbohydrate metabolism improving agent.
 8. Thescreening method described in claim 6 or claim 7, wherein the step ofmeasuring binding of (1) the polypeptide described in claim 1 or claim 2or a polypeptide consisting of an amino acid sequence having a homologyof 90% or more with the amino acid sequence represented by SEQ ID NO:2or SEQ ID NO:4, and which binds to Akt2, to (2) Akt2 is a step ofmeasuring a change in Akt2 based on the change in the aforementionedbinding.
 9. A method for producing a pharmaceutical composition forinsulin resistance improvement and/or carbohydrate metabolismimprovement, which comprises carrying out screening using the screeningmethod described in claim 6 to claim 8, and preparing a pharmaceuticalpreparation.